Fusion proteins and methods for treating HIV infection and AIDS related symptoms

ABSTRACT

Pharmaceutical compositions and methods for using a fusion protein having a superoxide dismutase and a transit peptide are described. Pharmaceutical compositions and methods for using the fusion protein in combination with other antiretroviral agents for treating patients with AIDS or HTV infection are also described.

REFERENCE TO SEQUENCE LISTING

Reference is made to the Sequence Listing in electronic format. TheSequence Listing is provided as a file entitled 15915568_1, created Jul.31, 2013, which is approximately 10.1 Kb in size. The information in theelectronic format of the Sequence Listing is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates generally to protein transduction domain(PTD) and superoxide dismutase fusion proteins and their use ininhibition of viral infection, and in particular infection of mammaliancells by the Human Immunodeficiency Virus (HIV) and in reduction orprevention of depletion of CD4+T cells as well as in improvement ofsymptoms and signs, quality of life.

BACKGROUND OF THE INVENTION

While human immunodeficiency virus (HIV) infection, which results inAIDS, is a relatively new infection in the human population, it hasquickly risen to the foremost health problem in the world. HIV/AIDS isnow the leading cause of death in sub-Saharan Africa, and is the fourthbiggest killer worldwide. While better treatment methods are now knownto prolong the life of patients with HIV infection, there is still nocure.

The HIV infection of CD4+ cells leads to progressive loss of CD4+ cells,causing the incidence of opportunistic infections and even death.Although there are divergent views on the pathogenesis of AIDS, one ofgenerally accepted views is that oxidative stress with excessive oxygenfree radicals caused by viral infection can activate the immune system(immune activation), and lead to progressive CD4+ cell depletion (Wanchuet al., 2009, AIDS Research and Human Retroviruses 25(12):1307-1311;Polyakov et al., Int Conf AIDS, 1992 Jul. 19-24; 8: 32 (abstract no. PuA6129)). Oxygen free radicals also reported to have relationship with HIVreplication (Oxidative stress and HIV infection: target pathways fornovel therapies? Future HIV Therapy, 2(4):327-338, 2008). Becausesuperoxide dismutase (SOD) is the most important antioxidant enzymewhich catalyzes the dismutation of superoxide anion free radical togenerate less toxic hydrogen peroxide and oxygen, it has been used totreat various oxygen free radical-related diseases (Winterbourn, 1993,Free Radical Biology & Medicine, 14:85-90). Studies indicated that SODgene expression was inhibited in HeLa cells which were infected with HIVtat gene, and reduced antioxidant enzyme defense leads to rapiddepletion of plasma thiol (Flores et al., Proc. Natl. Acad. Sci. USA,1993; 90; 7632-7636). It was found that lecithinized SOD (PC-SOD)inhibits human HIV types 1 and 2 in MT-4 cells (Premanathan, et al.,AIDS research and human retroviruses, 13(4), 1997).

Modern anti-HIV drugs target several different stages of the HIV lifecycle, and several of the enzymes that HIV requires to replicate andsurvive. Some of the commonly used anti-HIV drugs include nucleosidereverse transcriptase inhibitors such as ddI, AZT, d4T, 3TC andtenofovir; non-nucleoside reverse transcriptase inhibitors such asnevirapine, efavirenz and delavirdine; protease inhibitors such assaquinavir, amprenavir, lopinavir, ritonavir and indinavir. However, inmany HIV infected patients, none of these antiviral drugs, alone or incombination, is effective to prevent the progression of chronicinfection or treat acute AIDS. The high mutation rate of the HIV virusand associated emergence of HIV strains resistant to drugs is one largefactor that results in the inability to effectively treat HIV infection.Further, the modern anti-HIV drugs are expensive. In addition, themajority of currently available antiviral drugs of HIV showmitochondrial toxicity (Lewis, 1995, Mitochondrial toxicity of antiviraldrugs, Nature Med 1:417-422). For example, the mitochondrial toxicity ofAZT (zidovudine) results from mutation in mitochondrial DNA causedoxygen free radicals (Hayakawa et al., Biochem Biophys Res Commun, 1991,176:87-93).

Although oxygen free radicals were reported to be associated with HIVreplication and the destruction of CD4+ T cells, several clinical trailswith antioxidants indicated further trials were needed to determinewhether they had a beneficial effect in the treatment of HIV-infectedindividuals (Effect of N-acetylcysteine (NAC) treatment on HIV-1infection: a double-blind placebo-controlled trial, European Journal ofClinical Pharmacology, 50(6): 457-461, 1996; Improvement of immunefunctions in HIV infection by sulfur supplementation: Two randomizedtrials, Journal of Molecular Medicine, 78(1):55-62,2000; Restoration ofBlood Total Glutathione Status and Lymphocyte Function Followingα-Lipoic Acid Supplementation in Patients with HIV Infection, TheJournal of Alternative and Complementary Medicine, 14(2): 139-146,2008).

Accordingly, an inexpensive yet effective therapeutic and prophylactictreatment for HIV infection is desired. The treatment should be easilyadministered to those who are susceptible to or already have the virus.The present invention addresses these issues, as well as the problemspresented in the prior art.

SUMMARY OF THE INVENTION

The present invention relates to discovery of compositions and methodsof use of protein transduction domain (PTD) and superoxide dismutase(SOD) fusion proteins and optionally glutathione S-transferase (GST) aspart of the fusion protein, and their use to inhibit viral infections inmammalian cells, and in particular human HIV infection. Specifically,the present invention relates to compositions comprising the fusionproteins and methods to use these compositions to inhibit the growth ofHIV-1 infection in mammalian cells and patients.

Accordingly, in one aspect of the invention, a method for treating orinhibiting a Human Immunodeficiency Virus (HIV) infection, or treatingor inhibiting Acquired Human Immunodeficiency Syndrome (AIDS), in ahuman patient is provided. The method involves administering atherapeutically effective amount of a fusion protein, or a orpharmaceutical composition thereof, which contains a superoxidedismutase (SOD) and a transit peptide. The carboxy terminus of thetransit peptide is linked to the amino terminus of the SOD or viceversa, i.e., the amino terminus of the transit peptide is linked to thecarboxy terminus of the SOD. The human patient treated with the fusionprotein or pharmaceutical composition shows, as compared to theuntreated human, a decrease in HIV RNA production and CD4+T celldepletion as well as improvement of symptoms and signs, quality of life.The SOD can be an SOD containing copper and zinc (Cu—Zn-SOD), SODcontaining manganese (Mn-SOD) or an SOD containing iron (Fe-SOD). In oneembodiment, SOD is Cu—Zn-SOD. The transit peptide used is one with asequence of

(SEQ ID NO. 3) YGRKKRRQRRR, (SEQ ID NO. 4) YGRKKKKQRR, (SEQ ID NO. 5)YGRKKR, (SEQ ID NO. 6) RKKRRQRRR, (SEQ ID NO. 7) RRRRRRRRRand other polyarginine (with 5-30 amino acid residues),

(SEQ ID NO. 8) AAVALLPAVLLALLAP, (SEQ ID NO. 9) AVLLPVLLAAP,(SEQ ID NO. 10) VTVLALGALAGVGVG, (SEQ ID NO. 11) GALFLGFLGAAGSTMGA,(SEQ ID NO. 12) MGLGLHLLVLAAALQGAMGLGLHLLLAAALQGA, (SEQ ID NO. 13)WEAKLAKALAKALAKHLAKALAKALKACEA, (SEQ ID NO. 14)WEAALAEALAEALAEHLAEALAEALEALAA, (SEQ ID NO. 15)LARLLARLLARLLRALLRALLRAL, (SEQ ID NO. 16) KLLKLLLKLWKLLLKLLK,(SEQ ID NO. 17) RQIKIWFQRRMKKWK, (SEQ ID NO. 18)DAATATRGRSAASRPTERPRAPARSASRPRRPVE, (SEQ ID NO. 19)GWTLNSAGYLLGKINLKALAALAKKIL, (SEQ ID NO. 20) KETWWETWWTEWSQPKKKRKVor a polylysine (with 5-30 amino acid residues). The fusion protein canfurther include glutathione S-transferase (GST). In one embodiment, thecarboxy terminus of the GST is linked to the amino terminus of thetransit peptide. In a particularly preferred embodiment, the fusionprotein is PS1 with an amino acid sequence as set forth in FIG. 3 or PS2with an amino acid sequence as set forth in FIG. 4. The fusion proteinor the pharmaceutical composition containing the fusion protein can beadministered in pharmaceutically acceptable carrier by oral, buccal,sublingual, rectal or parenteral route.

In an embodiment, a therapeutically effective amount of at least oneother antiretroviral agent(s), including pharmaceutically acceptablesalts thereof, used for treatment of AIDS or HIV infection can beadministered in addition to the fusion protein. The at least one otherantiretroviral agent(s) can be nucleoside HIV reverse transcriptaseinhibitors, non-nucleoside HIV reverse transcriptase inhibitors and HIVprotease inhibitors. The dosage of the fusion protein can range from0.01-10.00 MU (million unit of SOD activity), the preferable range beingfrom 0.10-2.50 MU. The human patient may be resistant to antiretroviraltherapy (ART) and yet will show a decrease in HIV RNA production andCD4+T cell depletion.

In another aspect of the invention, a pharmaceutical compositioncontaining a glutathione S-transferase (GST), protein transductiondomain (PTD) and superoxide dismutase (SOD) fusion protein or just PTDand SOD fusion protein is provided. the fusion protein can be in apharmaceutically acceptable carrier. In one embodiment, thepharmaceutically acceptable carrier is appropriate for sublingualadministration. A kit containing the pharmaceutical composition is alsopart of the invention herein. In a preferred embodiment, the kitcontains either the fusion protein set forth in FIG. 3 or FIG. 4.

These and other aspects of the invention will be described in greaterdetail herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows schematics of various fusion proteins containingglutathione S-transferase (GST), protein transduction domain (PTD) andsuperoxide dismutase (SOD)

FIG. 2 shows schematics of various fusion proteins containing PTD andSOD

FIG. 3 shows amino acid sequence of GST-PTD-SOD fusion protein (SEQ IDNO. 1), referred to herein as PS1.

FIG. 4 shows amino acid sequence of SOD-PTD fusion protein (SEQ ID NO.2), referred to herein as PS2.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates generally to protein transduction domain(PTD) and superoxide dismutase (SOD) fusion proteins and their use ininhibition of viral infection, and in particular infection of mammaliancells by the Human Immunodeficiency Virus (HIV) and in reduction orprevention of depletion of CD4+T cells as well as in improvement ofsymptoms and signs, quality of life. This invention presents a class offusion proteins for inhibiting/treating of HIV infection, such fusionproteins containing at least one full-length SOD and at least one PTDand optionally glutathione S-transferase (GST). PTD is a protein domainwhich facilitates the efficient protein transduction across cellmembrane to transverse covalently linked proteins, DNA and othermolecules into almost all of tissues and cells, even across blood-brainbarrier with high transduction efficiency without damage to cells. Thedelivery of SOD enzyme across cell membrane can be successfully achievedby its fusion with PTD. It can efficiently remove the superoxide anionand thus the oxidative stress, protecting CD4+ cells from HIV andinhibiting HIV viral replication within the infected cells. These fusionproteins can effectively eliminate oxidative stress within both infectedcells and cells subject to risks of infection to achieve therapeuticeffects of reducing viral replication and raising CD4+ cell level.

Fusion proteins of the present invention (GST-PTD-SOD fusion protein andSOD-PTD fusion protein) can inhibit HIV infection, and can be useful forthe treatment of HIV infection and the consequent pathologicalconditions such as AIDS. As shown in the examples section below, thesefusion proteins are active on their own or in conjunction with a widevariety of other agents particularly those beneficial in HAART (highlyactive antiretroviral therapy) and other new combination compositionsand therapies.

One skilled in the art would know that there are three distinct types ofSOD depending on the metal cofactor in its reactive centre: SODcontaining copper and zinc (Cu—Zn-SOD); SOD containing manganese(Mn-SOD) or iron (Fe-SOD). The SOD contemplated in the present inventioncan be any of these SODs. Further, for example, it can be Cu/Zn-SOD ofhuman or bovine origin, can be recombinant, liposomal SODs as well asSODs of plant origin. Human Cu—Zn-SOD is preferred. Superoxide anionsgenerally form when molecular oxygen acquires an additional electron,which happens when oxygen is subjected to ionizing radiation. Superoxidedismutases (SODs) catalyze the dismutation of superoxide into oxygen andhydrogen peroxide.

Determination of dismutase activity of SOD can be madespectrophotometrically at 505 nm, one unit of SOD is expressed as theamount of protein causing a 50% inhibition of formazan dye (505 nm),employing xanthine and xanthine oxidase to generate superoxide radicals,at 37° C. and at pH 7.8, based on the method described by IrvinFridovich (Superoxide Radical: An Endogenous Toxicant, Annual Review ofPharmacology and Toxicology, Vol. 23: 239-257).

The present invention requires a PTD as part of fusion protein. PTD is aprotein domain which facilitates the efficient protein transductionacross cell membrane of covalently linked proteins (cargo proteins orpeptides or SODs), DNA and other molecules into almost all of tissuesand cells, even across blood-brain barrier with high transductionefficiency without damage to cells. The present invention relates tomethods and materials for delivering a SOD across cells and tissues andeven the blood-brain barrier into the brain. Delivery of SOD (cargoprotein) according to this invention is accomplished by the use of asuitable PTD (transit peptide or transduction domain). The transitpeptide allows for the transport of the C-terminus or N-terminus linkedor fused cargo (SOD) into mammalian cells, tissues and/or across theblood brain barrier. In one embodiment of the invention, an SOD islinked to a

(SEQ ID NO. 3) YGRKKRRQRRRtransit peptide of the invention. In another embodiment, the cargocompound is linked to a

(SEQ ID NO.4) YGRKKKKQRR or (SEQ ID NO. 5) YGRKKRtransport peptide of the invention. Other PTDs with amino acid sequences

(SEQ ID NO. 6) RKKRRQRRR, (SEQ ID NO. 7) RRRRRRRRR, (SEQ ID NO. 8)AAVALLPAVLLALLAP, (SEQ ID NO. 9) AVLLPVLLAAP, (SEQ ID NO. 10)VTVLALGALAGVGVG, (SEQ ID NO. 11) GALFLGFLGAAGSTMGA, (SEQ ID NO. 12)MGLGLHLLVLAAALQGAMGLGLHLLLAAALQGA, (SEQ ID NO. 13)WEAKLAKALAKALAKHLAKALAKALKACEA, (SEQ ID NO. 14)WEAALAEALAEALAEHLAEALAEALEALAA, (SEQ ID NO. 15)LARLLARLLARLLRALLRALLRAL, (SEQ ID NO. 16) KLLKLLLKLWKLLLKLLK,(SEQ ID NO. 17) RQIKIWFQRRMKKWK, (SEQ ID NO. 18)DAATATRGRSAASRPTERPRAPARSASRPRRPVE, (SEQ ID NO. 19)GWTLNSAGYLLGKINLKALAALAKKIL, (SEQ ID NO. 20) KETWWETWWTEWSQPKKKRKVor a polylysine (with 5-30 amino acid residues), or a polyarginine (with5-30 amino acid residues, e.g.,

(SEQ ID NO. 7) RRRRRRRRRcan also be successfully used as PTD in place of

(SEQ ID NO. 3) YGRKKRRQRRR, (SEQ ID NO. 4) YGRKKRRQRR or (SEQ ID NO. 5)YGRKKR.SOD may be linked to the C-terminus or N-terminus of the transitpeptide. In some embodiments, a given transit peptide is linked at itsN-terminus to glutathione S-transferase (GST). As used herein, the terms“linked,” “complexed” or “fused” refer to the chemical associationbetween protein or amino acid components being linked (as in GST-PTD-SODfusion protein or SOD-PTD fusion protein). In some cases, theassociation may not be direct, but may be mediated by a linking group oranother component. The chemical association between the components is bycovalent bonds. The fusion site or linkage site may be made susceptibleto enzymatic cleavage

The PTD may be nature, chemically synthesized or prepared by recombinanttechnology. Such natural synthetic or recombinant transit domains may beused to transport linked cargo into mammalian cells, tissues and/oracross the blood brain barrier. As used herein, the terms “polypeptide,”“peptide,” and “protein” are used interchangeably to refer to a polymerof amino acid residues. The terms apply to naturally occurring aminoacid polymers. The terms also apply to amino acid polymers in which oneor more amino acid residue is an artificial chemical analogue of acorresponding naturally occurring amino acid. The terms “polypeptide,”“peptide,” and “protein” are also inclusive of modifications including,but not limited to, glycosylation, lipid attachment, sulfation,gamma-carboxylation of glutamic acid residues, hydroxylation andADP-ribosylation. It will be appreciated that polypeptides are notalways entirely linear. For instance, polypeptides may be branched as aresult of ubiquitination and they may be circular (with or withoutbranching), generally as a result of post-translation events, includingnatural processing event and events brought about by human manipulationwhich do not occur naturally. Circular, branched and branched circularpolypeptides may be synthesized by non-translation natural process andby entirely synthetic methods as well. Specifically, these peptidesequences, which retain the inhibitory activity, can be preparedsynthetically in accordance with well known methods such as solid orsolution phase peptide synthesis. Alternatively, peptides of the presentinvention may be synthesized recombinantly.

For example, the fusion gene or nucleic acid sequence encodingGST-PTD-SOD fusion protein can be constructed by DNA recombinanttechnology and inserted into expression vector pGEX-2T. Suitablebacterial hosts such as E. coli (BL21, DH5α) are then transformed withthe expression vector pGEX-2T containing the inserted GST-PTD-SOD.pGEX-2T cloning vector, complete sequence (GenBank: U13850.1) (bothFASTA and graphics) (the sequence of which is incorporated herein byreference) can be obtained from the National Center for BiotechnologyInformation (NCBI), U.S. National Library of Medicine, 8600 RockvillePike, Bethesda Md., 20894 USA. The GST-PTD-SOD fusion protein can beexpressed in the E. coli by the induction IPTG and isolated by affinitychromatography for use in treating or inhibiting HIV infection/AIDS in ahuman patient.

The fusion gene or nucleic acid sequence encoding SOD-PTD can beconstructed by PCR technology and inserted into the chromosome of theyeast of Pichia pastoris via a shuttle vector such as any of pPICZα A, Bor C. Details of these vectors and vector sequences can be obtained fromInvitrogen Corporation, 1600 Faraday Ave. Carlsbad, Calif. 92008. Seealso Invitrogen User Manual, entitled EasySelect™ Pichia Expression KitFor Expression of Recombinant Proteins Using pPICZ and pPICZα in Pichiapastoris, Cat. No. K1740-01, Jun. 18, 2010, Manual Part No. 25-0172. Thevectors, pPICα A, B, and C, are 3.6 kb vectors used to express andsecrete recombinant proteins in Pichia pastoris. Recombinant proteinsare expressed as fusions to an N-terminal peptide encoding theSaccharomyces cerevisiae á-factor secretion signal. One skilled in theart would know that these vectors allow high-level, methanol inducibleexpression of the gene of interest in Pichia, and can be used in anyPichia strain including X-33, SMD1168H, and KM71H. These vectorstypically contain the following elements: AOX1 promoter for tightlyregulated, methanol-induced expression of the gene of interest; α-factorsecretion signal for directing secreted expression of the recombinantprotein; Zeocin resistance gene for selection in both E. coli andPichia; C-terminal peptide containing the c-myc epitope and apolyhistidine (6×His) tag for detection and purification of arecombinant fusion protein. These commercially available vectors (A, B,C versions) are provided with reading frames to facilitate in-framecloning with the C-terminal peptide. The fusion protein secreted by thehost cell (yeast or E. coli) can be purified by filtration, alcoholprecipitation and ion-exchange chromatography for use in treating orinhibiting HIV infection/AIDS in a human patient. “Patient” means ahuman infected with the HIV virus and in need of therapy as understoodby practitioners in the field of AIDS and HIV infection.

The active agent consisting of GST, a PTD and SOD fusion protein or aPTD and SOD fusion protein or compositions containing the active agentmay be administered to a human patient by any suitable route, forexample, by oral, buccal, sublingual, rectal or parenteral (includingintravenous, intramuscular, subcutaneous and intracoronaryadministration) route. Pharmaceutical compositions comprising thecompositions of the present invention can be formulated in aconventional manner using one or more physiologically acceptablecarriers or excipients or auxiliaries that facilitate processing of thecomposition, active agents or food additives, or a mixture thereof intopreparations which can be used therapeutically. In various embodiments,the composition includes carriers and excipients (including but notlimited to buffers, carbohydrates, mannitol, proteins, polypeptides oramino acids such as glycine, antioxidants, bacteriostats, chelatingagents, suspending agents, thickening agents and/or preservatives),water, oils, saline solutions, aqueous dextrose and glycerol solutions,other pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as buffering agents, tonicityadjusting agents, wetting agents and the like. It will be recognizedthat, while any suitable carrier known to those of ordinary skill in theart may be employed to administer the compositions of this invention,the type of carrier can vary depending on the mode of administration.

The fusion proteins of the invention will generally be given as apharmaceutical composition, and the active ingredient of the compositionmay be comprised of the fusion protein of the invention alone (i.e.,GST-PTD-SOD or SOD-PTD alone) or the fusion protein and at least oneother agent used for treating AIDS or HIV infection. The compositionswill generally be made with a pharmaceutically accepted carrier orvehicle, and may contain conventional excipients. The compositions aremade using common formulation techniques. The invention encompasses allconventional forms. Solid and liquid compositions are preferred. Somesolid forms include powders, tablets, capsules, and lozenges. Tabletsinclude chewable, buffered, and extended release. Capsules includeenteric coated and extended release capsules. Powders are for both oraluse and reconstitution into solution. Powders include lyophilizedpowders.

The compositions of the invention and pharmaceutical formulationsthereof can be administered in any amount effective to achieve itsintended purpose. When administrated to inhibit Human ImmunodeficiencyVirus (HIV) viral infection, treat AIDS and/or to reduce or preventdepletion of CD4+T cells in a host or host cell, the composition isadministered in a therapeutically effective amount. “Therapeuticallyeffective amount” means the amount of agent required to provide ameaningful patient benefit—suppression of viral load, suppression ofCD4+ T-cell depletion, restoration and preservation of immunologicfunction, improved quality of life, and/or reduction of HIV-relatedmorbidity and mortality. Guidance for the dosage and administrationschedule of various cargo compounds may be gathered from the manyreferences which describe the use of such compounds in treatment ordiagnosis, such as the Physicians' Desk Reference (PDR), or as otherwisedetermined by one of ordinary skill in the art.

The appropriate dosage may vary depending upon, for example, the humanpatient, the mode of administration and the nature and severity of thecondition being treated or diagnosed. However, in one embodiment of themethods of the present invention, satisfactory treatment results inhumans are indicated to be obtained at daily dosages from about 0.1 MUto about 2.5 MU (million unit of SOD activity) convenientlyadministered, for example, in daily doses, weekly doses, monthly doses,and/or continuous dosing. Daily doses can be in discrete dosages from 1to 12 times per day or more. Alternatively, doses can be administeredevery other day, every third day, every fourth day, every fifth day,every sixth day, every week, and similarly in day increments up to 30days or more. Dosing can be continuous, intermittent or a single dose,using any applicable dosing form, including tablet, saline, patches,i.v. administration and the like. More specifically, the composition isadministered in a therapeutically effective amount. While the inventionencompasses all conventional modes of administration as described above,oral, sublingual and rectal routes of administration are preferred. Thespecific dosing regime, however, will be determined by a physician usingsound medical judgment.

The invention also encompasses methods where a given fusion protein ofthe invention is given in combination therapy. That is, the given fusionprotein can be used in conjunction with, but separately from, otherantiretroviral agents useful in treating AIDS and HIV infection.“Combination,” “coadministration,” “concurrent,” and similar termsreferring to the administration of fusion protein of the invention withat least one other anti-HIV agent mean that the components (fusionproteins and other antiretroviral agents) are part of a combinationantiretroviral therapy or highly active antiretroviral therapy (HAART).HAART is well known to one skilled in the art. It uses a combination ofseveral antiretroviral drugs. The drugs inhibit the ability of the virusto multiply in the body, and they slow down the development of AIDS.HAART is defined as the use of three or more antiretroviral medications,one of which has to be a protease inhibitor or PI, an NNRTI ornon-nucleoside reverse transcriptase inhibitor, one of the nucleoside ornucleotide reverse transcriptase inhibitors or NRTIs (such as abacaviror tenofovir), an integrase inhibitor (e.g., raltegravir), or an entryinhibitor (e.g., Maraviroc or enfuvirtide) (WIHS publications, 2008).

The fusion proteins of this invention used in conjunction with otherantiretroviral agents or existing antiviral drugs is expected toeffectively remove oxygen free radicals within the mitochondria, play asynergistic therapeutic effect. Some of these other antiretroviralagents include HIV nucleoside reverse transcriptase inhibitors, such asddI, AZT, d4T, 3TC and tenofovir; HIV non-nucleoside reversetranscriptase inhibitors, such as nevirapine, efavirenz and delavirdine;HIV protease inhibitors, such as saquinavir, amprenavir, lopinavir,ritonavir and indinavir; HIV integrase inhibitor, such as raltegravir;HIV entry and fusion Inhibitors such as enfuvirtide and maraviroc. Theinvention, however, is not limited to these specific antiretroviralagents suitable for combination therapy. In these combination methods,GST-PTD-SOD or SOD-PTD will generally be given in a daily dose ofprotein with a SOD activity from 1000 U to 2,500,000 U in conjunctionwith other agents. These other antiretroviral agents generally will bepresent in a unit range similar to agents of that class used clinicallyor therapeutically. Typically, this is 0.25-1000 mg/unit. The specificdosing regime, however, will be determined by a physician using soundmedical judgment.

In one embodiment of the invention, a method for treating or inhibitinga Human Immunodeficiency Virus (HIV) infection, or treating orinhibiting Acquired Human Immunodeficiency Syndrome (AIDS), in a humanpatient involves the use of a fusion protein. Specifically, atherapeutically effective amount of a fusion protein containing asuperoxide dismutase (SOD) and at least one transit peptide with asequence of YGRKKRRQRRR (SEQ ID NO. 3) is administered. The carboxyterminus of the transit peptide is linked to the amino terminus of theSOD or vice versa. Cu—Zn-SOD is preferred. The fusion protein maycontain glutathione S-transferase (GST) linked to the amino terminus ofthe transit peptide. By administering the fusion protein, the humanpatient treated with the pharmaceutical composition shows, as comparedto the untreated human, a decrease in HIV RNA HIV production and CD4+Tcell depletion.

In other various embodiments of the present invention, the methods ofthe present invention may be used for diagnostic, prophylactic orresearch purposes or treatment ex vivo followed by return to the humanpatient in addition to the direct therapeutic procedures describedabove. In these embodiments, a fusion protein containing SOD isdelivered into a cell in vitro, ex vivo or in vivo. For example,delivery may be achieved in vitro by adding a GST, PTD and SOD fusionprotein or a PTD and SOD fusion protein to a cell culture, such as abiopsy. Alternatively, delivery may be achieved ex vivo by adding agiven fusion protein to a sample removed from a patient, for example,blood, tissue, or bone marrow, and returning the treated sample to thepatient.

WORKING EXAMPLES

The following working examples are provided to demonstrate preferredembodiments of the invention, but of course, should not be construed asin any way limiting the scope of the present invention. The examplesbelow were carried out using conventional techniques that are well knownand routine to those of skill in the art, except where otherwisedescribed in detail. Further, it should be appreciated by those of skillin the art that the techniques disclosed in the examples representtechniques found by the inventor to function well in the practice of theinvention, and thus can be considered to constitute preferred modes forits practice. However, those of skill in the art should, in light of thepresent disclosure, appreciate that many changes can be made in thespecific embodiments which are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention.

Example 1 SOD Containing Fusion Proteins and their Activity

Fusion proteins with and without GST were made, expressed and assayedfor its enzymatic or biological activity as follows:

Constructs preparation: The nucleic acid sequence encoding GST-PTD-SODfusion protein was constructed by DNA recombinant technology andinserted into expression vector pGEX-2T (GenBank: U13850.1). The fusiongene of SOD-PTD was constructed by PCR technology and inserted into thechromosome of the yeast of Pichia pastoris via the shuttle vector pPICZαA.

Cell culture and transfections: E. coli (BL21, DH5α) cells weretransformed with the expression vector pGEX-2T containing the insertedGST-PTD-SOD. The expression vector pPICZα A with the insertion ofSOD-PTD was transformed into Pichia pastoris (X-33).

GST-PTD-SOD fusion protein preparation: GST-PTD-SOD was expressed in theE. coli by the induction of IPTG and prepared by affinity chromatographyto electrophoretically pure for use.

SOD-PTD fusion protein preparation: SOD-PTD was secreted by the yeastafter induced by methanol; purified by filtration, alcohol precipitationand ion-exchange chromatography for use.

SOD assays: SOD activity was assayed spectrophotometrically at 505 nm.One unit was expressed as the amount of protein causing a 50% inhibitionof formazan dye (at 505 nm), employing xanthine and xanthine oxidase togenerate superoxide radicals, based on the method described by IrvinFridovich (Superoxide Radical: An Endogenous Toxicant, Annual Review ofPharmacology and Toxicology, Vol. 23: 239-257). SOD is Cu/Zn superoxidedismutase with a Gene ID of 6647 and NCBI Reference Sequence ofNP_000445.1. NCBI Reference (Protein_ID) for GST is AAA57089.1. Aminoacid sequence of GST-PTD-SOD (SEQ ID NO. 1):

SPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLVPRGS-YGRKKRRQRRR-ATKAVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSRKHGGPKDEERHVGDLGNVTADKDGVADVSIEDSVISLSGDHCIIGRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVIGIAQAmino acid sequence of SOD-PTD (SEQ ID NO. 2):

ATKAVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGEHVHEFGDNTAGCTSAGPHFNPLSRKHGGPKDEERHVGDLGNVTADKDGVADVSIEDSVISLSGDHCIIGRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVIG IAQ-YGRKKR

Example 2 Effect of Fusion Proteins PS1 and PS2 on HIV Viral Load andCD4+ T-Cell Count in HIV/AIDS Patients

PS1 is a fusion protein consisting of glutathione S-transferase (GST), aPTD with a sequence of

(SEQ ID NO. 3) YGRKKRRQRRRfused to the C-terminus of the GST, and Cu/Zn superoxide dismutase (SOD)fused to the C-terminus of the PTD as shown in FIG. 3. PS2 is a fusionprotein consisting of a PTD with a sequence of

(SEQ ID NO. 5) YGRKKRand Cu/Zn SOD fused to the N-terminus of the PTD as shown in FIG. 4. PS2does not consist of glutathione S-transferase (GST). These fusionproteins are simply referred to herein as PS1 and PS2 and should beunderstood as such. Recombinant Cu/Zn SOD was made together with the twofusion proteins (PS1 and PS2) according the procedures described inExample 1.

Rectal Mucosa Administration of PS1/PS2

A total of 11 volunteer HIV/AIDS male patients were recruited. Thesepatients were not subject to antiretroviral therapy (ART) or any otherantiviral therapy for at least 3 months before the treatment of thepresent invention. Their HIV-RNA count was >50 cp/mL, not associatedwith other major infectious diseases (such as N1H1-influenza,tuberculosis, etc.). These volunteer patients signed the clinical trialconsent after being well informed about PS1/PS2 by the attending doctor.They were randomly divided into four groups: (1) PS1 0.10 MU (millionunit of SOD activity) group; (2) PS2 0.10 MU group; (3) PS2 0.50 MUgroup; and (4) PS2 2.50 MU group. Patients' basic information in eachgroup is listed in Table 1-1. During the period of treatment, patientscontinued their work and normal life.

Pharmaceutical formulations, drug delivery, time, dose and duration: PS1and PS2 freeze-dried powder with designated dosages (SOD activities) wasdissolved into 2 ml saline and loaded to a 5-ml syringe with its needleremoved. The syringe was inserted 5-10 cm into the rectum through theanus, and solution was injected. Patients would lie prone on bed for10-30 min afterward. Each patient was administered PS1 or PS2 once everyday before sleeping. The total treatment period was one month. Bloodsamples were collected before and at the end of the treatment forlaboratory test. The laboratory analysis included CD4+ T-cell count andCD8+ T-cell count. CD8+ T cells kill HIV-infected cells, its count isassociated with viral load and HIV RNA load, and liver function, renalfunction, full blood cell count, glycemia and so on.

Clinical drug safety: During treatment, each group of HIV/AIDS patientsshowed no vomiting, blurred vision, limb joint pain, muscle pain,numbness, paresthesia and any other adverse reactions, except 1 patientin PS2 2.50 MU group developed mild fever, which disappeared afterappropriate rest. During the whole course of treatment, all patientsmaintained stable life symptoms, blood pressure, indicating soundclinical drug safety.

Changes in symptoms and signs: Compared with the medication before, eachgroup of patients with one month treatment demonstrated a ruddy facialcomplexion, a significant increase mental, physical energy and sexualdrive, and rash incidence dramatically decreased or stopped. No changeswere observed in appetite, diarrhea, body weight.

CD4+ T-cell count and viral load changes: Patients' CD4+ T-cell countand viral load changes are shown in Table 1-2.

With one month of the administration of PS1, the patient's CD4+ wasincreased by 37.6/μL while the effect on the viral load decrease wasmodest. With one month of the administration of PS2, viral load wasdecreased with groups of lower dosages (0.10 MU and 0.50 MU), whilemaximum increase in CD4+ cell count was observed with 0.50 MU group. Thehigh dosage group of 2.50 MU showed the least efficacy in both viralload reduction and CD4+ increase. Virus load changes of +0.19 for PS1and −0.15 for PS2 2.50 MU are all within the error tolerance range sincethe detection method's 95% reliable error allowance is 0.3 Lg;

Rectal mucosa administration of PS1/PS2 had no discernable side effectson HIV/AIDS patients and can significantly improve symptoms and signs,improve their quality of life.

Sublingual Administration of PS1

A total of 10 volunteer HIV/AIDS patients were recruited. These patientswere not subject to antiretroviral therapy (ART) or any other antiviraltherapy for at least 3 months prior to the treatment described herein.These patients were paid blood donors infected with HIV in 1994-1996.Their HIV-RNA count was >50 cp/mL, not associated with other majorinfectious diseases (such as N1H1-influenza, tuberculosis, etc.). Thesevolunteer patients signed the clinical trial consent after being wellinformed about PS1. They were randomly divided into two groups:treatment group and control group, patients' basic information in eachgroup is listed in Table 1-3. During the period of treatment, allselected patients received centralized management but acted freely. Thetreatment began after a week of life to adapt to the environment.

Pharmaceutical formulations: Tablets were used in this experiment. Thetablets for the treatment group contained food additives and 2,500U/tablet of PS1, while the tablet for the control group contained foodadditives only. These tablets were manufactured in a food processingplant.

Drug delivery: Each patient was sublingually administered with PS1tablets or control tablets.

Delivery time, dosage and duration: there times every day on AM 8:00, PM2:00 and PM 8:00, each time 16 tablets. The total treatment period was 3months.

Blood samples were collected pre-treatment and each one monthpost-treatment for laboratory test.

The laboratory analysis included CD4+ T-cell count and CD8+ T-cellcount, HIV RNA load and liver function, renal function, full blood cellcount, glycemia, and so on.

Clinical drug safety: During treatment, each group of HIV/AIDS patientsshowed no vomiting, blurred vision, limb joint pain, muscle pain,numbness, paresthesia and any other adverse reactions, except 1 patientin control group developed mild nausea once during the whole course oftreatment. During medication, 3 patients in treatment group and 1patient in control group developed fever associated with mild dizzinessand headache; which disappeared after appropriate rest.

Patients showed no liver and kidney function deterioration after threemonths of sublingual administration of the tablets, and maintainedstable life symptoms and blood pressure indicating sound clinical drugsafety. Clinical symptoms and signs of patients before and aftermedication were shown in Table 1-4.

The patients in the treatment group with three-month medication showedruddy facial complexion, and their mental, physical energy and malesexual drive were significantly increased, while hair loss wassignificantly decreased and 3 subjects grew new hair. Their body weightincreased 4.34 Kg compared with 2.50 Kg of the control group; their rashincidence decreased or stopped, appetite, diarrhea did not changesignificantly. The patients in control group didn't show any improvementin their looking, mental and physical energy, hair loss, skin rash,diarrhea, appetite, male sexual function and other symptoms and signs.

CD4+ T-cell count and viral load changes: Patients' CD4+ T-cell countand viral load changes were shown in are shown in Table 1-5.

Sublingual administration of PS1 tablets for 3 months (three timesdaily, every time 16 tablets, each tablet of 2,500 U) demonstrated agood antiviral effect in HIV/AIDS patients. Compared with the controlgroup, the viral load in the treatment group decreased by 1.08 LOG 10.The administration of PS1 also improved immune function. CD4+ T-cellcount showed increase, but not as significantly.

Sublingual administration of PS1 showed no obvious side effects onHIV/AIDS patients and can significantly improve their complexion, mentaland physical energy, hair, skin rashes, sexual function and othersymptoms and signs, improve their quality of life.

Example 3 Effect of Fusion Protein PS1 on HIV Viral Load and CD4+ T-CellCount in HIV/AIDS Patients Resistant to Antiretroviral Therapy (ART)

A total of 10 volunteers were recruited. They were paid blood donorsinfected with HIV in 1994-1996. These HIV/AIDS patients had regulartreatment of ART for at least 3 months prior to the treatment describedfurther herein. Their HIV-RNA count was >50 cp/mL, not associated withother major infectious diseases (such as N1H1-influenza, tuberculosis,etc.). These volunteer patients signed the clinical trial consent afterbeing well informed about PS1. They were randomly divided into twogroups: treatment group and control group, patients' basic informationin each group is listed in Table 2-1. During the period of treatment,all selected patients received centralized management but acted freely.The treatment began after a week of life to adapt to the environment.

Pharmaceutical formulations: Tablets were used in this experiment. Thetablets for the treatment group contained food additives and 2,500U/tablet of PS1, the tablet for the control group contained foodadditives only, these tablets were manufactured in a food processingplant.

Drug delivery: Each patient was sublingually administered with PS1tablets or control tablets. Delivery time, dosage and duration: Theretimes every day at AM 8:00, PM 2:00 and PM 8:00, each time 16 tablets.The total treatment period was 3 months. Blood samples were collectedpre-treatment and each one month post-treatment for laboratory test. Thelaboratory analysis included CD4+ T-cell count and CD8+ T-cell count,HIV RNA load and liver function, renal function, full blood cell count,glycemia, and so on.

Clinical drug safety: During the treatment, each group of HIV/AIDSpatients showed no vomiting, blurred vision, limb joint pain, musclepain, numbness, paresthesia and any other adverse reactions except the 2patients in the treatment group and the 2 patient in the control groupdeveloped mild nausea once. During medication, 4 patients in thetreatment group and 3 patients in the control group developed feverassociated with mild dizziness and headache, which disappeared afterappropriate rest.

During the whole course of medication, patients showed no liver andkidney function deterioration, kept with stable life symptoms, bloodpressure, indicating a sound clinical drug safety. Changes in symptomsand signs: Clinical symptoms and signs of patients before and aftermedication were shown in Table 2-2.

Patients in treatment group demonstrated ruddy facial complexion,significant increase in mental, physical energy strength and sexualdrive, and a significant decrease in hair loss with 2 subjects growingnew hairs. Their body weight increased 3.33 Kg compared with 2.43 Kg ofthe control group; Appetite, rash, diarrhea did not changesignificantly. No significant changes were observed with the patients inthe control group in their looking, spirit, physical strength, hairloss, skin rash, diarrhea, appetite, male sexual function and othersymptoms and signs. CD4+ T-cell count and viral load changes: Patients'CD4+ T-cell count and viral load changes are shown in Table 2-3.

Sublingual administration of PS1 tablets (three times daily, every time16 tablets, each tablet 2,500 U) demonstrated a remarkable antiviraleffect in HIV/AIDS patients resistant to ART (also referred to herein asHIV/AIDS resistant patients). Compared with the control group, the viralload in the treated group decreased by 1.27 LOG 10, more than 1 LOG 10.The administration of PS1 also improved immune function with an increaseof CD4+ T-cell count.

Sublingual administration of PS1 had no discernable side effects onHIV/AIDS patients and could significantly improve their complexion,mental, physical strength, hair, skin rashes, sexual function and othersymptoms and signs, improve their quality of life.

Example 4 Effect of Rectal Administration of Fusion Protein PS2 inCombination with Highly Active Antiretroviral Therapies (HARRT) on HIVViral Load and CD4+ T-Cell Count in HIV/AIDS Patients

A total of 3 volunteer HIV/AIDS male patients with treatment of HAART(AZT+3TC+EFV) for at least 3 months before this study were recruited.Their viral load was lower than detective level and not associated withother major infectious diseases (such as N1H1-influenza, tuberculosis,etc.). These volunteer patients signed the clinical trial consent afterbeing well informed about PS2. They were randomly divided into 3 groups:PS2 0.10 MU (million unit of enzymatic activity) group, PS2 0.50 MUgroup, PS2 2.50 MU group. Patients' basic information in each group islisted in Table 3-1. During the period of treatment, patients continuedtheir work and normal life.

Pharmaceutical formulations, drug delivery, time, dosage and duration:freeze-dried powder of PS2 of the corresponding dosage was dissolvedinto 2 ml saline and then loaded into a 5-ml syringe with the needleremoved. The syringe was inserted 5-10 cm into the rectum through theanus, and the solution was injected. The patient lay prone on bed for10-30 min afterwards. Each patient was administered PS2 once every daybefore sleeping. The total treatment period was one month. Blood sampleswere collected pre-treatment and post-treatment for laboratory test. Thelaboratory analysis included CD4+ T-cell count and CD8+ T-cell count,HIV RNA load and liver function, renal function, full blood cell count,glycemia, and so on.

Clinical drug safety: During treatment, each group of HIV/AIDS patientsshowed no vomiting, blurred vision, limb joint pain, muscle pain,numbness, paresthesia and other adverse reactions except 1 patient inPS2 2.50 MU group developed mild fever, which disappeared afterappropriate rest; during whole course of treatment, all patientsmaintained stable life symptoms, blood pressure, indicating a soundclinical drug safety. Changes in symptoms and signs: each group ofpatients with one month treatment demonstrated ruddy complexion,significant improved in mental, physical strength significantlyincreased and appetite improved.

CD4+ T-cell count and viral load changes: Patients' CD4+ T-cell countchange is shown in Table 3-2. Their viral load kept in LDL (lowerdetective level).

Within one month of the treatment, the administration of PS2 showedsignificant improvement in immune function. The CD4+ T-cell countincreased by 81.7, an average increase of 31.6%. The effect in the casesof the dosage of 0.50 million and 2.50 million was better. Meanwhile,the administration of PS2 did not interfere with antiviral effect ofHARRT. Rectal mucosa administration of PS2 combined with HARRTsignificantly improved symptoms and signs of HIV/AIDS patients andimprove their quality of life.

TABLE 1-1 Basic information of clinical trial cases of HIV/AIDS Averageduration of Group Case Average age (years) HIV infection (years) PS10.10 MU 2 23.50 ± 3.54 2.00 ± 1.41 PS2 0.10 MU 3 29.33 ± 1.15 2.33 ±0.59 PS2 0.50 MU 3 30.67 ± 3.79 2.00 ± 1.00 PS2 2.50 MU 3 32.00 ± 7.001.67 ± 1.15

TABLE 1-2 The effect of rectal administration of PS1/PS2 on CD4+ T-cellcount and viral load of HIV/AIDS Patients^(a) Average CD4 cell count(/μL) Average viral load (Log10) Group Before After Change Before AfterChange PS1 0.10 MU 376.0 413.6 +37.6 4.41 4.60 +0.19 PS2 0.10 MU 342.7343.9 +1.2 4.44 3.96 −0.48 PS2 0.50 MU 644.7 679.4 +34.7 3.95 3.51 −0.44PS2 2.50 MU 256.7 209.1 −47.6 5.09 4.94 −0.15 ^(a)CD4+ T-cell count andHIV RNA load: tested in National Centre for AIDS Prevention and Control(NCAIDS) of CDC, China. CD4+ T-cell count: Utilization ofCD3/CD8/CD45/CD4 antibody, TruCOUNT tubes, FACS lysing solution, (BDBiosciences, San Jose, California, USA). HIV RNA load: NucliSens HIV-1QT, NASBA, bioMe'rieux (Marcy L'Etoile, France).

TABLE 1-3 Basic information of clinical trial cases of HIV/AIDS SexAverage age Average duration of Group Case Male Female (years) HIVinfection (years) Treatment 8 6 2 45.88 ± 3.40 13~15 Control 2 1 1 46.00± 1.6  13~15

TABLE 1-4 Clinical symptoms and signs of patients before and aftermedication Complexion Spirit Physical strength Group Before After BeforeAfter Before After Treatment  1.38 ± 0.74 0.38 ± 0.52** 0.75 ± 0.71 0**1.00 ± 0.76   0** Control 1.00 ± 0   1.00 ± 0      0 0   0 0 AppetiteHair Diarrhea Group Before After Before After Before After Treatment0.50 ± 0.93 0.25 ± 0.46 1.13 ± 0.95 0* 1.00 ± 0 0.63 ± 0.52 Control 0 00.50 ± 0.71 0.50 ± 0.71 1.00 ± 0 0.50 ± 0.71 Rash Sexual function (male)Weight Group Before After Before After Before After Treatment 0.25 ±0.46 0 2.17 ± 0.75 0.50 ± 0.55** 62.79 ± 9.04  67.13 ± 8.64  Control 0 00.50 ± 0.71 0.50 ± 0.71   80.00 ± 16.97 82.50 ± 17.68 Note:, * and **stand for significant difference with P < 0.05 and P < 0.01,respectively, when comparing the differences in the symptoms and signsof the same group before and after medication.

TABLE 1-5 The effect of sublingual administration of PS1 on CD4+ T-cellcount and viral load of HIV/AIDS patients^(a) Viral load (Log10) ChangeCD4+ T-cell count (/μL) After After After after After After After Changeone two three three one two three after three Group Before month monthsmonths months Before month months months months Treatment 4.84 4.67 4.174.28 −0.56 306 301 248 313 7(2.3%) 72 Control 5.05 5.22 5.21 5.57 0.52281 313 323 353 72 (25.6%)

TABLE 2-1 Basic information of clinical trial cases of HIV/AIDSresistant patients Sex Average age Average duration of Group Case MaleFemale (years) HIV infection (years) Treatment 6 4 2 51.50 ± 9.48 13~15Control 4 3 1 50.75 ± 1.26 13~15

TABLE 2-2 Clinical symptoms and signs of patients before and aftermedication Complexion Spirit Physical strength Group Before After BeforeAfter Before After Treatment 1.50 ± 0.55 0.33 ± 0.52** 0.83 ± 0.41 0**1.33 ± 0.52 0.17 ± 0.41** Control 1.25 ± 0.50 1.25 ± 0.50   0.75 ± 0.960.50 ± 0.58 0.75 ± 0.96 0.75 ± 0.96   Appetite Hair Diarrhea GroupBefore After Before After Before After Treatment 0.33 ± 0.82 0.17 ± 0.410.83 ± 0.75 0* 0.67 ± 0.52 0.67 ± 0.52 Control 0.25 ± 0.50 0.50 ± 1.000.75 ± 0.50 0.75 ± 0.50 0.75 ± 0.50 0.75 ± 0.50 Rash Sexual function(male) Weight Group Before After Before After Before After Treatment 0 02.75 ± 0.50  1.50 ± 1.01* 64.42 ± 9.66 67.75 ± 9.36 Control 0 0.25 ±0.50 2.33 ± 0.58 2.33 ± 0.58 58.95 ± 2.73 61.38 ± 2.87 Note:, * and **stand for significant difference with P < 0.05 and P < 0.01,respectively, when comparing the differences in the symptoms and signsof the same group before and after medication..

TABLE 2-3 The effect of sublingual administration of PS1 on CD4+ T-cellcount and viral load of HIV/AIDS resistant patients^(a) Viral load(Log10) CD4+ T-cell count (CD4/μL) After After After Change After AfterAfter Change one two three after three one two three after three GroupBefore month months months months Before month months months monthsTreatment 4.02 3.80 3.93 3.59 −0.43 358 283 294 366 8   (2.2%) Control4.02 5.10 4.62 4.86 0.84 200 155 132 156 −44 (−22.0%)

TABLE 3-1 Basic information of clinical trial cases of HIV/AIDS Durationof HIV Group Case Average age (years) infection (years) PS2 0.10 MU 1 459 PS2 0.50 MU 1 40 6 PS2 2.50 MU 1 30 4

TABLE 3-2 The effect of rectal mucosa administration of PS2 combinedwith HARRT on CD4+ T-cell count of HIV/AIDS patients ^(a) CD4+ T-cellcount (/μL) Average Group Before After Change Change 0.10 MU 250 310  60(24.0%) 0.50 MU 340 458 118 (34.7%) 81.7 (31.6%) 2.50 MU 187 254  67(35.8%) ^(a) CD4+ T-cell count: tested in National Centre for AIDSPrevention and Control (NCAIDS) of CDC, China. CD4+ T-cell count:Utilization of CD3/CD8/CD45/CD4 antibody, TruCOUNT tubes, FACS lysingsolution, (BD Biosciences, San Jose, California, USA).

Thus, it has been demonstrated herein that the fusion proteins of thepresent invention are able to inhibit HIV activity and effective fortreating HIV infected patients. The invention, however, should not belimited to the fusion proteins exemplified above. In fact, it may proveto be the case that the most useful pharmacological small moleculepeptides and nucleic acids designed and synthesized in light of thisdisclosure will be second generation derivatives of the exemplifiedmolecules.

All publications mentioned in the specification are indicative of thelevel of those skilled in the art to which this invention pertains. Allpublications, and patents and patent applications, if any, are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference. Although the foregoinginvention has been described in some detail by way of illustration andexample for purposes of clarity of understanding, it will be obviousthat certain changes and modifications may be practiced within the scopeof the appended claims.

What is claimed is:
 1. A fusion protein comprising the amino acidsequence of SEQ ID NO:
 2. 2. A pharmaceutical composition comprising thefusion protein of claim 1 in a pharmaceutically acceptable carrier.
 3. Akit comprising the pharmaceutical composition of claim
 2. 4. A methodfor treating or inhibiting a Human Immunodeficiency Virus (HIV)infection, or treating or inhibiting Acquired Human ImmunodeficiencySyndrome (AIDS), in a human patient, comprising: administering to thehuman patient a therapeutically effective amount of a fusion protein, ora pharmaceutical composition thereof, wherein the fusion proteincomprises the amino acid sequence of SEQ ID NO:
 2. 5. The method ofclaim 4, wherein the human patient treated with the fusion protein orpharmaceutical composition shows, as compared to the untreated humanpatient, a decrease in HIV RNA production and CD4⁻ T cell depletion. 6.The method of claim 4, wherein the fusion protein or the pharmaceuticalcomposition thereof is administered to the human patient in apharmaceutically acceptable carrier by an oral, buccal, sublingual,rectal or parenteral route.
 7. The method of claim 4 further comprisingadministering a therapeutically effective amount of at least one otherantiretroviral agent used for treatment of AIDS or HIV infectionselected from the group consisting of nucleoside HIV reversetranscriptase inhibitors, non-nucleoside HIV reverse transcriptaseinhibitors and HIV protease inhibitors, wherein the dosage range of thefusion protein is 0.01-10.00 MU (million units of superoxide dismutase(SOD) activity).
 8. The method of claim 4, wherein the human patient isresistant to antiretroviral therapy (ART).
 9. The method of claim 4,wherein the fusion protein, or the pharmaceutical composition thereof isadministrated together with highly active antiretroviral therapy(HAART).